In mammalian central nervous system dopamine transporter (DAT) represents the primary mechanism for clearance of dopamine (DA) from the extracellular space. DAT expression is regulated long-term by chronic drug administration; it is also regulated in a rapid, dynamic fashion by many factors including DAT substrates. To date attempts to address the rapid DAT regulation mechanisms have been made mostly in non-neuronal cell models. These studies demonstrated that the short-term DAT regulation occurs by altered transporter trafficking, and thereby cell surface expression. However, the proper analysis of DAT transport and endocytosis in neuronal cells is complicated with current techniques. To understand how the dynamic constitutive and drug-induced DAT regulation occurs in the brain, how it contributes to normal neurotransmission and how it plays a role in the addictive properties of stimulants, we need to develop new methods that will allow analysis of DAT regulation in neuronal model systems. We suggest development of a novel imaging technique to study DAT dynamics in single cultured neurons with new photoactivatable fluorescent proteins (PAFPs). Since protein tagged with a photoactivatable label will fluoresce only upon the local photo switching, it therefore can be visualized and tracked in a spatially- and temporally-defined manner. This imaging technique will allow for the analysis of kinetics and mechanisms of subcellular transport of a newly synthesized DAT to axonal varicosities and dendrites (Aim 1), and to determine dynamics of internalization and recycling of the DAT in axons and somatodendritic compartments (Aim 2). We will also apply two distinct PAFPs to analyze regulation of the DAT trafficking by physical or signal mediated interactions with D2 presynaptic receptors (Aim 3). Furthermore, we will extend common fluorescence resonance energy transfer (FRET) method to a photoactivatable FRET to detect association within a limited pool of the pulse-chase photolabeled DAT and D2 molecules in a living neuron. The kinetic characteristics of DAT trafficking to different compartments will be measured, and the respective critical targeting residues will be determined. The distinct subcellular steps of DAT regulation through internalization and recycling and the quantitative difference between these mechanisms will be studied. The imaging technologies being developed and the experiments proposed in this project will allow us to better appreciate contribution of DAT to normal neurotransmission and may result in new strategies for drug abuse prevention and treatment. [unreadable] [unreadable] [unreadable] [unreadable]